Recent findings indicate that epigenetics and the fetal environment play a critical part in the adult onset and progression of familial diseases. However, it is not known whether in utero exposure of phthalates, ubiquitous environmental toxins, has epigenetic effects that are inherited to the next generation. Phthalates are known to cause testicular dysgenesis syndrome, characterized by postnatal anomalies such as increased crytorchidism, shortened anogenital distance, aberrant spermatogenesis, and testicular cancer, stemming from impaired fetal testicular development. In our preliminary studies, we found that di(2- ethylhexyl) phthalate (DEHP), the most abundant phthalate, caused disorganization of germ cells in seminiferous tubules, changes in the distribution of spermatogenic stages, increased apoptosis, increased number of multinucleated gonocytes, and decreased sperm counts, which are collectively indicative of impaired spermatogenesis. Interestingly, these effects were observed in the mouse testis of F1 to F4 generation (F1-F4) offspring of DEHP-treated F0 dams. Our preliminary results also showed a novel finding that these testicular transgenerational effects are in part originating from impaired function of spermatogonial stem cells (SSCs). Moreover, the comparison of DNA methylation patterns of sperm DNA from F3 offspring of DEHP- and vehicle oil-treated F0 dams showed a number of significantly strong differentially methylated regions, indicating that an altered DNA methylation may be one of the underlying epigenetic mechanisms for the transgenerational effects. Thus, we seek to test the hypothesis that exposing F1 germ cells to DEHP in utero causes transgenerational effects in a dose-dependent manner, and that this transgenerational outcome -- including impairment of spermatogonial stem cell function -- is due to alterations in the DNA methylation pattern in the germline that persists in subsequent generations. We plan to determine (1) dose-dependent transgenerational effects on the testis; (2) if altered spermatogonial stem cell (SSC) potential is the underlying cellular mechanism for transgenerational effects; and (3) if altered DNA methylation pattern is the underlying epigenetic mechanism of transgenerational effects. This dose-dependent study, in combination with studies to determine the cellular and molecular mechanisms, is the best way to evaluate if transgenerational effects of phthalates are possible for animals and humans and what mechanisms are responsible for these transgenerational effects. Completion of this proposal has the potential to impact our thinking on the fetal exposure of environmental toxicants and heritable human diseases, and the role of phthalates on targetting the SSC-related genes, altering the SSC proliferation and differentiation programming.